Technical Field
The present disclosure relates to a method for enhancing the efficiency of nucleic acid amplification. More particularly, the present disclosure relates to a device used in polymerase chain reaction (PCR), which is for enhancing the efficiency of nucleic acid amplification by controlling the thermal convection flow rate of the buffer in the tube to increase the single thermal convection circulation period.
Description of Related Art
For genetic research such as genetics and molecular biology or the detection of animal and plant diseases, it needs to amplify a few copies of nucleic acids by nucleic acid amplification, such as polymerase chain reaction (PCR), from a small amount of nucleic acid samples to the amount which can be detected in a short time. The nucleic acid amplification products can be further hybridized to nucleic acid probes conjugated with fluorescent, radioactive substances or colorimetric enzyme, thus producing fluorescence, radiation image or color reaction.
The aforementioned process of the polymerase chain reaction includes three main steps: denaturation, annealing reaction and extension reaction, and the required reaction temperature of the three main steps are different. Nowadays the sample of commercialized PCR amplification technology includes the template DNA far amplification, the oligonucleotide primers which are complementary to particular sequences of each strand of template DNA, thermostable DNA polymerase, and deoxy-ribonucleoside triphosphate (dNTP), then the reaction repeats heating and cooling the sample, and the sample is circulated between different temperatures to amplify the particular part of template DNA nucleic acid sequence.
In the aforementioned denaturation, the sample is heated to high temperatures, causing the double-stranded template DNA to be separated into single-stranded DNA; in the annealing reaction, the sample is cooled to a lower temperature, allowing annealing of the primers to the single-stranded DNA templates which are formed in denaturation to form a DNA-primer hybrid; in the extension reaction, the sample is maintained at an appropriate temperature, and the primer of the DNA-primer hybrid can be extended to synthesize a new DNA strand complementary to each of the DNA template through the action of DNA polymerase; the DNA sequences between the binding sites of the forward primer and the reverse primer can be replicated in each cycle consisting of three main steps.
The temperature of the sample tube of conventional PCR reaction apparatus is controlled by heat conduction, for example, the PCR sample tube is in contact with a solid metal block of high thermal conductivity, changing the temperature of the sample tube by heating and cooling to reach the required temperature of the PCR sample tube. The majority of sample tubes are held in a vertical position during heating, and the metal block with thermal conductivity is disposed at the bottom of the sample tube to generate temperature difference in the tube to generate thermal convection circulation phenomenon in the internal buffer. However, the effectiveness of the nucleic acid amplification will be influenced by the thermal convection velocity of buffer. When the thermal convection velocity is too fast, it will cause the single thermal convection circulation period to be less than the single nucleic acid amplification period, resulting in the nucleic acid amplification to be incomplete. By contrast, when the thermal convection velocity is too slow, it will increase the required spent time of nucleic acid amplification. The flow rate of the buffer will be influenced by multiple factors, such as the compositions of the buffer solution and the temperature differences between the top of the tube and the bottom of the tube. Therefore, in order to change the flow rate of the buffer, the currently practiced manners include adding substances into the buffer to change the viscosity of buffer, or controlling the temperature differences between the top of the tube and the bottom of the tube. Although these manners can indeed change the thermal convection rate, the thermal convection rate is often not effectively manipulated due to these manners are very difficult to control.